Project Summary: Hematopoietic stem cells (HSCs) are a rare population of cells characterized by their ability for life-long self- renewal and differentiation to all blood cell lineages. HSC function is regulated by complex cell-intrinsic and cell-extrinsic pathways, but the mechanisms that control HSC differentiation and self-renewal are not fully understood. Elucidating the regulation of HSCs is critical to improving therapies for hematopoietic recovery after stress, such as during bone marrow transplantation. This proposal aims to investigate the regulation of HSCs by N6-methyladenosine (m6A). This reversible RNA modification of adenosine is abundant throughout the transcriptome of eukaryotic cells and functions as an epitranscriptomic regulator of target RNA processing, but, to our knowledge, no study has yet investigated the role of m6A in HSCs. Our preliminary evidence suggests that m6A is preferentially required in HSCs compared to other hematopoietic cells. We found that loss of m6A led to increases in HSC numbers, but decreases HSC function. This proposal seeks to explore m6A-mediated RNA regulation in vivo in HSCs biology through both cellular and molecular approaches. I will determine the role of m6A in regulating HSC cell fate and functional capacity for the first time. I will also use a novel methylated RNA sequencing method for low cell numbers to characterize the transcriptome-wide m6A profile of HSCs and uncover HSC-specific m6A targets. I will then determine how m6A impacts target processing in HSCs and what effects that has on regulating HSC biology. To our knowledge, this study is the first to define the role of m6A RNA methylation in HSC regulation. The results of this proposal will uncover a novel layer of regulation on HSC function and potentially reveal novel ways to improving HSC function.